Transgene-free genome editing in marine algae by bacterial conjugation - comparison with biolistic CRISPR/Cas9 transformation.

The CRISPR/Cas9 technology has opened the possibility for targeted genome editing in various organisms including diatom model organisms. One standard method for delivery of vectors to diatom cells is by biolistic particle bombardment. Recently delivery by conjugation was added to the tool-box. An important difference between these methods is that biolistic transformation results in transgene integration of vector DNA into the algae genome, whereas conjugative transformation allows the vector to be maintained as an episome in the recipient cells. In this study, we have used both transformation methods to deliver the CRISPR/Cas9 system to the marine diatom Phaeodactylum tricornutum aiming to induce mutations in a common target gene. This allowed us to compare the two CRISPR/Cas9 delivery systems with regard to mutation efficiency, and to assess potential problems connected to constitutive expression of Cas9. We found that the percentage of CRISPR-induced targeted biallelic mutations are similar for both methods, but an extended growth period might be needed to induce biallelic mutations when the CRISPR/Cas9 system is episomal. Independent of the CRISPR/Cas9 vector system, constitutive expression of Cas9 can cause re-editing of mutant lines with small indels. Complications associated with the biolistic transformation system like the permanent and random integration of foreign DNA into the host genome and unstable mutant lines caused by constitutive expression of Cas9 can be avoided using the episomal CRISPR/Cas9 system. The episomal vector can be eliminated from the diatom cells by removal of selection pressure, resulting in transient Cas9 expression and non-transgenic mutant lines. Depending on legislation, such lines might be considered as non-GMOs.

'Myrosin cells' are not a prerequisite for aphid feeding on oilseed rape (Brassica napus) but affect host plant preferences.

The enzyme myrosinase (EC 3.2.3.1.147) is present in specialised myrosin cells and forms part of the glucosinolate-myrosinase system, also known as 'the mustard oil bomb', which has an important role in the defence system of cruciferous plants against insect pests. Transgenic Brassica napus MINELESS have been produced by transgenic ablation of myrosin cells. This prompted us to investigate the importance of myrosin cells in plant-aphid interactions. In order to study this, we challenged transgenic MINELESS and wild-type cultivar Westar seedlings with the aphids Brevicoryne brassicae (a specialist) and Myzus persicae (a generalist). Our study included aphid free-choice and aphid fecundity experiments. Data from these experiments showed that B. brassicae prefers wild-type seedlings and M. persicae prefers MINELESS. B. brassicae and M. persicae showed significant variation in establishment on plants regardless of whether they were wild type or MINELESS and also differed significantly in affecting plant parts. Myrosinase activity in MINELESS control seedlings was 83.6% lower than the wild-type control seedlings. Infestation with either of the two aphid species induced myrosinase levels in both wild-type and MINELESS seedlings. Infestation with M. persicae reduced the concentration of most glucosinolates while B. brassicae had the opposite effect. B. brassicae enhanced the formation of glucosinolate hydrolysis products both in wild-type and MINELESS seedlings. However, M. persicae decreased All ITC but increased 3,4ETBut NIT in wild-type seedlings. Taken together, the investigation shows that the presence of myrosin cells affects the preference of generalist and specialist aphid species for Brassica napus plants.

Ecotype dependent expression and alternative splicing of epithiospecifier protein (ESP) in Arabidopsis thaliana.

Epithiospecifier protein (ESP) is responsible for diverting glucosinolate hydrolysis from the generation of isothiocyanates to that of epithionitriles or nitriles, and thereby negatively affects the ability of the plant to defend itself against certain insects. Despite this important role of ESP, little is known about its expression in plant tissues and the regulation thereof. We therefore investigated ESP expression by qPCR and Western blot in different organs during the growth cycle of the two Arabidopsis thaliana ecotypes Col-0 and Mt-0. Besides the fact that ESP transcript and protein levels were revealed to be much higher in Mt-0 than in Col-0 in all cases, our qPCR results also indicated that ESP expression is regulated differently in the two A. thaliana ecotypes. No ESP protein was detected by Western blot in any organ or developmental stage for Col-0. During the assays an alternative splice variant of ESP was identified in Col-0, but not Mt-0, leading to a mis-spliced transcript which could explain the low expression levels of ESP in the former ecotype. Analysis of genomic sequences containing the ESP splice sites, of ESP protein level and ESP activity from seven A. thaliana ecotypes showed a positive correlation between the presence of a non-canonical 5' splice site for ESP and the absence of detectable ESP protein levels and ESP activity. When analysing the expression of both transcript variants in Col-0 after treatment with methyl jasmonate, a condition known to "induce ESP", it was indeed the alternative splice variant that was preferentially induced.

The synthesis and enzymic hydrolysis of (E)-2-[2,3-2H2]propenyl glucosinolate: confirmation of the rearrangement of the thiohydroximate moiety.

(E)-2-[2,3-2H2]propenyl glucosinolate was synthesised starting from (E)-[3,4-2H2]but-3-en-1-ol, which was produced by reduction of but-3-yn-1-ol with deuterium gas in the presence of Lindlar's catalyst. The synthesis of (E)-2-[2,3-2H2]propenyl glucosinolate was completed via the nitro intermediate to form the basic desulphoglucosinolate skeleton. The (E)-2-[2,3-2H2]propenyl glucosinolate was fully characterised and deuterium NMR spectroscopy used to examine the rearrangement of the thiohydroximate to the isothiocyanate and thiocyanate.

Characterization and evolution of a myrosinase from the cabbage aphid Brevicoryne brassicae.

The aphid myrosinase gene has been elucidated using Rapid Amplification of cDNA Ends-PCR. Sequencing has shown that aphid myrosinase has significant sequence similarity (35%) to plant myrosinases and other members of glycosyl hydrolase family 1 (GHF1). The residues acting as proton donor and nucleophile, in the hydrolysis of glucosinolates by aphid myrosinase, are identified as Glu 167 and Glu 374 respectively. The equivalent residues in plant myrosinase are Gln 187 and Glu 409 and for the cyanogenic beta-glucosidase Glu 183 and Glu 397. Thus it would appear that the absence of a proton donor is not necessary for the hydrolysis of glucosinolates as was thought to be the case for the plant myrosinases. Aphid myrosinase appears to be more similar to animal beta-O-glucosidases than to plant myrosinases, as assessed by sequence similarity and phylogenetic techniques. These results strongly suggest that myrosinase activity has twice arisen from beta-O-glucosidases in plants and animals. Comparison of aphid myrosinase with plant myrosinase has highlighted Lys 173 and Arg 312 as possibly playing a crucial role in the hydrolysis of glucosinolates by aphid myrosinase.

Microautoradiographic localisation of a glucosinolate precursor to specific cells in Brassica napus L. embryos indicates a separate transport pathway into myrosin cells.

The in-situ localisation of a desulpho-glucosinolate precursor has been studied by microautoradiography of cryo-sections from immature seeds and pods of the high-glucosinolate Brassica napus L. cv. Argentine collected 23 days after pollination. After feeding with the tritium-labelled glucosinolate precursor [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid, embryo radicles, cotyledons and pod-wall were frozen in liquid nitrogen. Cryotome sections were freeze-dried and coated with nuclear emulsion autoradiographic film. A distinct pattern of radioactivity derived from the glucosinolate precursor was found in specific cells in both radicle and cotyledons. In contrast, the labelling in pod walls was not cell specific, but general at the inner side of the pod wall. The results show that the glucosinolate/desulphoglucosinolate was localised in specific cells, in a pattern resembling that of myrosin cells known to contain myrosinase (EC 3.2.3.1). In addition [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to immature seeds and pods of B. napus and a quantitative incorporation into 2-hydroxy-3-butenylglucosinolate and 3-butenyl-glucosinolate was observed. When [4,5-3H](beta-D-glucopyranosyl)-4-pentenethiohydroxamic acid was fed to 4-day-old seedlings the label was taken up by all tissues. We propose a model in which glucosinolate/desulphoglucosinolates are transported to myrosin cells to participate in the myrosinase-glucosinolate multifunctional defence system.

Purification and characterisation of a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.).

Plant myrosinases and glucosinolates constitute a defence system in cruciferous plants towards pests and diseases. We have purified for the first time a non-plant myrosinase from the cabbage aphid Brevicoryne brassicae (L.) to homogeneity. The protein was N-terminally blocked and protease (trypsin and lys c) degradation gave peptides of which five were sequenced. The protein is a dimer with subunits of mass 54 kDa+/-500 Da. Western blot analysis with an anti-aphid myrosinase antibody showed a strong cross reaction with a protein extract from the Brassica specialist, B. brassicae. The anti-aphid myrosinase antibody does not cross react with plant myrosinase neither does an anti-plant myrosinase antibody cross react with aphid myrosinase.

Genetic structure and evolution of RAC-GTPases in Arabidopsis thaliana.

Rho GTPases regulate a number of important cellular functions in eukaryotes, such as organization of the cytoskeleton, stress-induced signal transduction, cell death, cell growth, and differentiation. We have conducted an extensive screening, characterization, and analysis of genes belonging to the Ras superfamily of GTPases in land plants (embryophyta) and found that the Rho family is composed mainly of proteins with homology to RAC-like proteins in terrestrial plants. Here we present the genomic and cDNA sequences of the RAC gene family from the plant Arabidopsis thaliana. On the basis of amino acid alignments and genomic structure comparison of the corresponding genes, the 11 encoded AtRAC proteins can be divided into two distinct groups of which one group apparently has evolved only in vascular plants. Our phylogenetic analysis suggests that the plant RAC genes underwent a rapid evolution and diversification prior to the emergence of the embryophyta, creating a group that is distinct from rac/cdc42 genes in other eukaryotes. In embryophyta, RAC genes have later undergone an expansion through numerous large gene duplications. Five of these RAC duplications in Arabidopsis thaliana are reported here. We also present an hypothesis suggesting that the characteristic RAC proteins in higher plants have evolved to compensate the loss of RAS proteins.

Purification and characterisation of epithiospecifier protein from Brassica napus: enzymic intramolecular sulphur addition within alkenyl thiohydroximates derived from alkenyl glucosinolate hydrolysis.

Epithiospecifier protein (ESP), a ferrous ion dependent protein, has a potential role in regulating the release of elemental sulphur, nitriles, isothiocyanates and cyanoepithioalkanes from glucosinolates. Two classes of ESP polypeptides were purified with molecular masses of 39 and 35 kDa, and we show that the previously reported instability was conditionally dependent. The 39 kDa polypeptide was made up of two distinct isozymes (5.00, 5.14) whilst several were present for the 35 kDa form of ESP (5.40-5.66). An anti-ESP antibody reacted with both the 39 and 35 kDa ESP forms in Brassica napus and strongly with a polypeptide corresponding to the 35 kDa ESP form in Crambe abyssinica, but did not detect any ESP in Sinapis alba or Raphanus sativus. A cytochrome P-450 mediated iron dependent epoxidation type mechanism is suggested for ESP.

Horizontal gene transfer from transgenic plants to terrestrial bacteria--a rare event?

Today, 12 years after the first field release of a genetically modified plant (GMP), over 15,000 field trials at different locations have been performed. As new and unique characteristics are frequently introduced into GMPs, risk assessment has to be performed to assess their ecological impact. The possibilities of horizontal gene transfer (HGT; no parent-to-offspring transfer of genes) from plants to microorganisms are frequently evaluated in such risk assessments of GMPs before release into the field. In this review we indicate why putative HGT from plants to terrestrial (soil and plant associated) bacteria has raised concern in biosafety evaluations. Further, we discuss possible pathways of HGT from plants to bacteria, outline the barriers to HGT in bacteria, describe the strategies used to investigate HGT from plants to bacteria and summarize the results obtained. Only a few cases of HGT from eukaryotes such as plants to bacteria have been reported to date. These cases have been ascertained after comparison of DNA sequences between plants and bacteria. Although experimental approaches in both field and laboratory studies have not been able to confirm the occurrence of such HGT to naturally occurring bacteria, recently two studies have shown transfer of marker genes from plants to bacteria based on homologous recombination. The few examples of HGT indicated by DNA sequence comparisons suggest that the frequencies of evolutionarily successful HGT from plants to bacteria may be extremely low. However, this inference is based on a small number of experimental studies and indications found in the literature. Transfer frequencies should not be confounded with the likelihood of environmental implications, since the frequency of HGT is probably only marginally important compared with the selective force acting on the outcome. Attention should therefore be focused on enhancing the understanding of selection processes in natural environments. Only an accurate understanding of these selective events will allow the prediction of possible consequences of novel genes following their introduction into open environments.

Natural transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms.

A small microcosm, based on optimized in vitro transformation conditions, was used to study the ecological factors affecting the transformation of Acinetobacter calcoaceticus BD413 in soil. The transforming DNA used was A. calcoaceticus homologous chromosomal DNA with an inserted gene cassette containing a kanamycin resistance gene, nptII. The effects of soil type (silt loam or loamy sand), bacterial cell density, time of residence of A. calcoaceticus or of DNA in soil before transformation, transformation period, and nutrient input were investigated. There were clear inhibitory effects of the soil matrix on transformation and DNA availability. A. calcoaceticus cells reached stationary phase and lost the ability to be transformed shortly after introduction into sterile soil. The use of an initially small number of A. calcoaceticus cells and nutrients, resulting in bacterial growth, enhanced transformation frequencies within a limited period. The availability of introduced DNA for transformation of A. calcoaceticus cells disappeared within a few hours in soil. Differences in transformation frequencies between soils were found; A. calcoaceticus cells were transformed at a higher rate and for a longer period in a silt loam than in a loamy sand. Physical separation of DNA and A. calcoaceticus cells had a negative effect on transformation. Transformation was also detected in nonsterile soil microcosms, albeit only in the presence of added nutrients and at a reduced frequency. These results suggest that chromosomal DNA released into soil rapidly becomes unavailable for transformation of A. calcoaceticus. In addition, strain BD413 quickly loses the ability to receive, stabilize, and/or express exogenous DNA after introduction into soil.

Cloning and characterization of rac-like cDNAs from Arabidopsis thaliana.

The Rho family of GTPases are in higher eukaryotes divided into 3 major subfamilies; the Rho, Rac and Cdc42 proteins. In plants, however, the Rho family is restricted to one large family of Rac-like proteins. From work with mammalian phagocytes the Rac proteins are known to activate a multicomponent NADPH-dependent oxidase which results in accumulation of H2O2, a process termed oxidative burst. In plants a similar oxidative burst is observed and plays and important role in its defence against pathogen infections, suggesting a similar role for the plant Rac-like proteins. The Rho family of GTPases proteins are also involved in control of cell morphology, and are also thought to mediate signals from cell membrane receptors. In a broad search for members of the Ras superfamily in plants, several new small GTP-binding proteins were found. We report here the identification and molecular cloning of 5 rac-like cDNAs from Arabidopsis thaliana, Arac1-5. The Rac-like proteins deduced from the cDNA sequences all share 80-95% homology, but show considerably more diversity on the nucleotide level, indicating that this is an ancient gene family. Four of the rac genes were found to be expressed in all tissues examined, but one gene, Arac2, was expressed exclusively in the root, hypocotyl and stem. Our results show that the rac gene family in A. thaliana consists of at least 10 different genes.

Induced Natural Transformation of Acinetobacter calcoaceticus in Soil Microcosms.

Factors affecting natural transformation of Acinetobacter calcoaceticus BD413 with homologous chromosomal DNA in a silt loam soil microcosm were investigated. Inducible transformation of declining populations of noncompetent A. calcoaceticus cells was detectable for up to 6 days when a simple carbon source, salts, and freshly added DNA were used. In two different experimental setups, the residence time in soil of induced cells could be increased to either 11 or 24 h before DNA addition without reduced transformation frequency; 200-to 1,000-fold fewer transformants were observed following the addition of water. These observations suggest that A. calcoaceticus remains transformable for several hours after its activation by nutrients in soil. Increasing the levels of phosphate salts significantly enhanced the numbers of transformants without increasing the recipient counts correspondingly. Variable levels of ammonium or divalent cations (Mg(sup2+) and Ca(sup2+)) did not have a similar major influence. Soil moisture content significantly affected the transformation frequency of A. calcoaceticus cells, with a general tendency of higher frequencies in drier soil. A minimal frequency was observed at around 35% soil moisture. The data indicate that A. calcoaceticus cells in soil which cannot be detectably transformed are easily induced by nutrients to undergo natural transformation with chromosomal DNA. Access to nutrients seems to be critical for the development and maintenance of competence in soil, which is also affected by abiotic factors like moisture level and phosphate salt concentration.